During the past five year period, this Merit Award has permitted us to focus on molecular ?epigenomic? strategies that combinatorially regulate programs of gene transcription. These include identifying new epigenetic regulators; defining signatures of active enhancers; and providing evidence that enhancers mediating actions of liganded nuclear receptors are themselves regulated transcription units, transcribing functionally important eRNAs. We have also identified new histone modifications and elucidated their biological functions, and contributed to the growing evidence for dynamic three-dimensional interaction/reorganization of specific genomic regions. Finally, we have provided initial evidence that lncRNAs could exert their biological functions based on their actions to allosterically modify functions of RNA-binding proteins, and of retinoic acid receptor regulation of Pol III-dependent Alu repeat transcription units and their roles in biological processes. Specific contributions include: elucidating the roles of PHF8, PHF2 and SMYD5 in transcriptional regulation; identifying roles of a tissue-specific splice variant of LSD1; finding a novel tyrosine phosphorylation of H2A and; demonstrating the role of JMJD6 demethylase as a regulator of pause-release by actions on specific enhancers. We characterized the E2-regulated enhancer program and the ligand-dependent increase in eRNA transcription as a signature of functional enhancers. We identified the recruitment of the MegaTrans complex and Condensins I and II to these functional enhancers. Using Pit1 as a model, we also demonstrated the requirement for Pit1-dependent enhancer interactions with the Matrin3-rich network for effective activation of homeodomain-dependent transcriptional programs in the endocrine system. We propose to continue our work in this extension by exploring the roles of nuclear receptors in regulating negative, as well as positive, enhancer-dependent transcriptional programs, defining the roles of JMJD6 in these programs, and roles of modified long-distance interactions in dictating the chromosomal alterations underlying regulated transcriptional response. We will use genetic approaches to elucidate the mechanisms of super-enhancer regulation of endocrine developmental programs in the endocrine system. RELEVANCE (See instructions): Under this MERIT Award, we have contributed to understanding the enhancer-dependent strategies regulating broad gene transcriptional programs. We will continue and extend these studies in the MERIT Extension period by investigating global enhancer interaction networks; mechanisms of negative as well as positive gene transcriptional regulatory programs; and elucidating developmental roles of component enhancers in superenhancers in the endocrine system.